Method and system for buoyant separation

ABSTRACT

A method and system for buoyant separation of a target constituent of a sample, the method comprising: at a process chamber, combining a volume of substrates having a first density with the sample, thereby producing a population of target-bound complexes comprising the target constituent bound to at least a portion of the volume of substrates; within the process chamber, physically separating the population of target-bound complexes from the sample based upon interaction between the volume of substrates and an applied force; aggregating the population of target-bound complexes at a collection region of the process chamber; extracting the population of target-bound complexes from the process chamber; and processing the target constituent from the population of target-bound complexes for further analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/004,874, filed 11 Jun. 2018, which is a continuation-in-part of U.S.application Ser. No. 14/969,446, filed 15 Dec. 2015, which claims thebenefit of U.S. Provisional Application Ser. No. 62/092,019, filed on 15Dec. 2014 and U.S. Provisional Application Ser. No. 62/189,518 filed on7 Jul. 2015, which are each incorporated herein in their entirety bythis reference. This application also claims the benefit of U.S.Provisional Application No. 62/616,647 filed on 12 Jan. 2018 and62/517,543 filed on 9 Jun. 2017, which are both incorporated herein intheir entirety by this reference.

TECHNICAL FIELD

This invention relates generally to separation methods and systems inthe fields of biological sample processing. More specifically, itrelates to an improved method and system for buoyant separation ofparticles in a sample of biological fluid.

BACKGROUND

In research and diagnostic applications, it is often important to beable to isolate one or more types of particles of a sample. Isolation oftarget components in an efficient and high throughput manner can thushave a significant impact in healthcare applications, biologicalresearch, research in the food industry, and medical research.Components for isolation and extraction can include cells, proteins,nucleic acids, lipids, and other particles commonly found in biologicalfluid, and in one example, efficient isolation of rare cancerous cells(e.g., circulating tumor cells) in a biological sample can be used todetect and/or diagnose cancer for a patient at an early stage whereintervention is critical. There are several conventional setups used forparticle isolation from samples, implementing techniques derived fromone or more of: fluorescence activated sorting, magnetic sorting,filtration, electrophoretic separation, and other methods of separation.However, conventional particle isolation systems are typicallyinefficient, are not high-throughput, are labor intensive, are prone touser-error, and require large systems, necessitating a significantamount of training, and/or contributing to untrustworthy analyses.Conventional setups are also typically expensive to operate, from time,labor, and cost perspectives, which can provide limits upon thecompleteness of an analysis performed using such setups.

Thus, there is a need in the biological sample processing field tocreate an improved method and system for buoyant separation of targetcomponents of a sample. This invention provides such an improved methodand system.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are flowchart schematics of embodiments of a method forbuoyant separation of a target constituent of a sample.

FIG. 1C depicts an optical micrograph of a variation of substrates usedin an embodiment of a method for buoyant separation of a targetconstituent of a sample.

FIG. 1D depicts an example of a block of an embodiment of a method forbuoyant separation of a target constituent of a sample.

FIGS. 1E-1I depict an example of a block of an embodiment of a methodfor buoyant separation of a target constituent of a sample.

FIG. 1J depicts a specific example of a process chamber used inembodiments of a system and method for buoyant separation of a targetconstituent of a sample.

FIGS. 1K-1M depict a specific example of a block of an embodiment of amethod for buoyant separation of a target constituent of a sample.

FIGS. 2A-2C depict variations of interactions between sample fluid and aprocess chamber in an embodiment of a method for buoyant separation of atarget constituent of a sample.

FIG. 2D depicts specific examples of variations of interactions betweensample fluid and a process chamber in embodiments of a method forbuoyant separation of a target constituent of a sample.

FIGS. 3A-3D depict variations of a process chamber morphologicalconfigurations in embodiments of a system and method for buoyantseparation of a target constituent of a sample.

FIG. 4 depicts a specific example of a process chamber used in anembodiment of a method for buoyant separation of a target constituent ofa sample.

FIG. 5 depicts a specific example of a process chamber used in anembodiment of a method for buoyant separation of a target constituent ofa sample.

FIGS. 6A-6B depict variations of collection regions, dependent uponrotation axis, in an embodiment of a method for buoyant separation of atarget constituent of a sample.

FIGS. 7A-7B depict a variation of extraction in an embodiment of amethod for buoyant separation of a target constituent of a sample.

FIG. 8 depicts a variation of extraction in an embodiment of a methodfor buoyant separation of a target constituent of a sample.

FIG. 9A-9B depict variations of a process chamber comprising a detectionwindow in an embodiment of a method for buoyant separation of a targetconstituent of a sample.

FIG. 10 depicts a variation of a method for substrate modification in amethod for buoyant separation of a target constituent of a sample.

FIG. 11 depicts an embodiment of a system for buoyant separation of atarget constituent of a sample.

FIGS. 12A and 12B depict examples of process chambers used in a methodfor buoyant separation of a target constituent of a sample.

FIG. 13 depicts an example of a process chamber for buoyant separationof a target constituent of a sample.

FIG. 14 depicts an embodiment of a system for buoyant separation of atarget constituent of a sample.

FIGS. 15A-15D depict a variation of an embodiment of a system forbuoyant separation of a target constituent of a sample.

FIG. 15E depicts an example related to the first variation of anembodiment of a system for buoyant separation of a target constituent ofa sample.

FIGS. 16A-16F depict a variation of an embodiment of a system forbuoyant separation of a target constituent of a sample.

FIGS. 17A-17C depict a variation of an embodiment of a system forbuoyant separation of a target constituent of a sample.

FIGS. 18A-18B depict an alternative embodiment of a system for buoyantseparation of a target constituent of a sample.

FIGS. 19A-19C depict an alternative embodiment of a system for buoyantseparation of a target constituent of a sample.

FIGS. 20A-20B depict variations of a subcomponent of a process chamberused in an embodiment of a system for buoyant separation of a targetconstituent of a sample.

FIG. 20C depicts a variation of a subcomponent of a process chamber usedin an embodiment of a system for buoyant separation of a targetconstituent of a sample.

FIG. 21A-21B depict examples of an embodiment of a system for buoyantseparation of a target constituent of a sample.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of preferred embodiments and examples of theinvention is not intended to limit the invention to these preferredembodiments and examples, but rather to enable any person skilled in theart to make and use this invention.

1. Method

As shown in FIGS. 1A and 1B, an embodiment of a method 100 for buoyantseparation of a target constituent of a sample comprises: at a processchamber, combining a volume of substrates having a first density withthe sample, thereby producing a population of target-bound complexescomprising the target constituent bound to at least a portion of thevolume of substrates Silo; within the process chamber, physicallyseparating the population of target-bound complexes from the samplebased upon interaction between the volume of substrates and an appliedforce S120; aggregating the population of target-bound complexes at acollection region of the process chamber S130; extracting the populationof target-bound complexes from the process chamber S140; and processingthe target constituent from the population of target-bound complexes forfurther analysis S150. However, the method 100 can additionally oralternatively be implemented in any other suitable steps, such as in amanner analogous to U.S. application Ser. No. 14/969,446, filed 15 Dec.2015, which is incorporated in its entirety by this reference.

The method 100 functions to provide a process for efficiently separatingtarget constituents from a sample with a low amount of effort fromresearch or clinical personnel. The method 100 also functions tofacilitate downstream analyses (e.g., polymerase chain reaction-basedassays, lateral flow assays, culture-based assays, etc.) of isolated andcollected target constituents, for research applications, clinicalapplications, and/or food industry applications. Preferably, the method100 can be applied to a large sample volume (e.g., 35 mL sample volumes,sample volumes over 5 mL, etc.), in order to extract one or more targetcomponents from the large sample volume. However, the method 100 canadditionally or alternatively be applied to any other suitable sizedsample volume (e.g., sample volumes smaller than or equal to 5 mL). Themethod 100 preferably utilizes passively applied forces (e.g.,gravitational force) and/or actively applied forces (e.g., centrifugalforce) to separate target constituents from a bulk sample based upondensity differences between bound target constituents and the bulksample; however, the method 100 can additionally or alternatively useany other physical parameter and associated separation mechanism toenhance isolation of a target constituent from a biological sample. Forinstance, in one variation, buoyant separation can be enhanced withmagnetic manipulation of buoyant particles, by coupling a targetconstituent to particles having buoyant and magnetic-dual functionality.Upon separation of the target constituent from the sample, extraction ofthe target constituent can be performed according to the method 100.Additionally and/or alternatively, non-target constituents can be boundto buoyant substrates and removed according to the method 100. In othervariations, a non-target constituent of the sample can be tagged withmagnetic substrates and drawn or repelled to a desired region of aprocess chamber (e.g., wall of the process chamber), while the targetconstituent that has been tagged with buoyant substrates is separatedfrom the sample volume. However, any other suitable mode of separationprocess can be implemented in addition to or in substitution of theseparation methods described.

Furthermore, the method 100 can be used to simultaneously orsequentially separate each of a set of target constituents from a bulksample, based upon selective coupling of each of the set of targetconstituents to an associated substrate volume that can be individuallyisolated based upon a feature (e.g., physical feature, chemical feature,etc.). In variations, the method 100 can be used for separation oftarget cell and/or target analyte constituents from a biological sample,by using buoyant particles that facilitate concentration of the targetconstituents to a collection location (e.g., an extremum most opposite agravitational or centrifugal force). The method 100 is preferablyimplemented, at least in part, using elements of the system 200described in Section 2 below; however, the method 100 can additionallyor alternatively be implemented using any other suitable system.

In a specific example, the method 100 can be used to providebuoyancy-activated separation and/or extraction of target constituentsof a sample from a bulk sample volume of 1 Liter within 30 minutes. In asecond specific example, the method 100 can be used to providebuoyancy-activated separation and/or extraction of target constituentsof a sample from a bulk sample volume of 50 mL within 15 minutes.However, variations of the method 100 can alternatively be tuned toenable separation according to any other suitable timeline.

1.1 Method—Tagging with Buoyant Substrates

Block S110 recites: at a process chamber, combining a volume ofsubstrates having a first density with the sample, thereby producing apopulation of target-bound complexes comprising the target constituentbound to at least a portion of the volume of substrates. Block S110functions to provide interaction between the target constituent and thevolume of substrates, thereby enabling selective manipulation of thetarget constituent in subsequent blocks of the method 100. Block S110 ispreferably implemented using an embodiment, variation, and/or example ofthe process chamber described in Section 2 below; however, Block S110can additionally or alternatively be implemented using any othersuitable process chamber(s) for combination of a sample with a volume ofa process material. Furthermore, variations of Block S110 can includereceiving a sample volume having a population of target-bound complexes,where combination and/or complexification was performed in a separateprocess chamber. Embodiments, variations, and/or examples of Block S110are further described in U.S. application Ser. No. 14/969,446, filed 15Dec. 2015.

In Block S110 substrates of the volume of substrates preferably comprisesubstrate particles (e.g., beads, spheres, micelles, microbubbles) andcan include any one or more of: plastic beads (e.g., polypropylenebeads, polyethylene beads, etc.), glass beads, lipid beads (e.g.,stabilized liposome-based beads), hollow beads, solid beads,liquid-filled beads, and any other suitable type of particle.Furthermore, while substrates used in Block S110 are preferablysubstrate particles, Block S110 can additionally or alternativelyinclude using planar or non-planar substrates (e.g., plates, surfaces,surface coatings, etc.), or substrates having any other suitablemorphology that facilitates separation of the target constituent fromthe sample.

The substrates are preferably characterized by a first density lowerthan that of the density (i.e., a second density) of fluid of the sample(e.g., ranging from 0.1 g/cm³ and 0.99 g/cm³). As such, substrates ofthe volume of substrates are preferably configured to float within thesample to facilitate separation in subsequent blocks of the method 100(e.g., buoyant particles). However, substrates of the volume ofsubstrates can alternatively be configured with any other suitabledensity relative to that of the density of fluid and/or untargetedconstituents of the sample to facilitate separation.

In examples, the substrates have a diameter from 10 nm to 100 nm intargeting analytes or 1 μm to 30 μm in targeting cells; however, theparticles can have any other suitable dimension configured facilitateefficient binding with elements of the target constituent.

In one variation, the volume of substrates comprises silica beads havinga density less than that of fluid of the sample, wherein the silicabeads are treated with a moiety (e.g., Streptavidin for biotin binding,an antibody for formation of an antibody-antigen complex, anothermoiety, etc.) configured to selectively couple with associated portionsof the target constituent (e.g., cell, analyte) of the sample. In asecond variation, the volume of substrates comprises microbubbles (e.g.,gas-filled microparticles, hollow microspheres, colloidal bubbles) thatcan be spheroidal, skirted, ellipsoidal or any other suitablethree-dimensional shape. The shape of the microbubbles can varydynamically in response to the fluid dynamics of the sample volume(e.g., changing from one shape to another dictated by gravity,viscosity, and surface tension), but can alternatively be a fixed shape.In a specific example, as shown in FIG. 1C, the microbubbles arecomprised of borosilicate glass that can include a particle shellsurrounding a particle core (e.g., gas filled, fluid-filled,particle-filled, etc.). However, the particle shell can be alternativelycomposed of any other suitable material including lipids, proteins,surfactants, polymers, and/or any suitable combination thereof. In thisexample, the glass microbubbles can be fabricated with a fixedspheroidal shape defining a particle diameter (e.g., ranging frombetween 5 to 30 micron), and a particle shell thickness (e.g., less than2 micron thick). In solution, the volume of microbubbles to volumetarget constituent (e.g., target cell) ratio preferably ranges between1:2 to 5:1. However, the substrates can be of any other suitablecomposition, shape, density, and/or dimension.

Furthermore, the substrates can be configured with moieties for bindingto the target constituent (e.g., red blood cells, white blood cells,T-cells, circulating tumor cells, stem cells, etc.) and can additionallyor alternatively include any one or more of: charge-based moieties,nucleic acid-targeting moieties, protein-based moieties (e.g., celladhesion molecules, growth factors, synthetic proteins), and any othersuitable moiety. In a specific example, the particle shell of the glassmicrobubbles can be coated with an aminosilane layer to allow forsubsequent surface functionalization with biomolecules (e.g.,antibodies, aptamers, lectins, oligos, etc.). After glass microbubbleshave been amino-functionalized, the glass microbubbles are preferablycrosslinked to streptavidin. However, any other suitable chemicalprocedure can be performed for surface functionalization of thesubstrates (e.g., PEGylation, click chemistry, layer-by-layer assembly,ink-jet printing etc.) for selective capture of target constituents,using any other suitable moiety.

In another variation, substrates can additionally and/or alternativelyfunction as a signal delivery agent to target constituents (e.g., via arecombinant molecule bound to the surface of the substrate particle). Ina specific example, CD3+ T cells can be captured using a microbubbledisplaying Cd28, a protein which can stimulate the T cell (e.g.,inducing cell proliferation and cytokine production), a primary step tomanufacturing T cells expressing a chimeric antigen receptor (e.g.,CAR-T cells) used in cell therapy (e.g., cancer treatment). However, thesubstrates can be otherwise configured with any other suitable moietyfor multifunctional applications including target-bound complexseparation and extraction.

Preferably, combining the volume of substrates (substrate volume) withthe sample containing the target constituent (target sample volume) inBlock S110 is performed in a manner that provides sufficient combinationand dwell time to achieve sufficient complexification (i.e., forming ofcomplexes) of the target constituent to the substrate particle (e.g.,with a desired binding efficiency). In solution (e.g., PBS, water, RPMI,etc.), the initial concentration of the substrates within the volume ofsubstrates, prior to combination of the substrate volume and the targetsample volume, is preferably 40 to 125 million substrate particles permL, but can be any other suitable concentration. The complexificationbinding efficacy can be dependent on the volume ratio of buoyantparticles to target constituent in the total sample volume (combinedsample volume). In a specific example, complexification with a bindingefficiency of 90% can occur in a buffer solution that includes at leastone of PBS, EDTA, and BSA wherein the volume ratio of buoyant particlesto target constituent is 1:2, with a dwell time of 20 minutes. In asecond specific example, complexification with a binding efficiency ofup to 98% binding efficiency can occur in a buffer solution thatincludes at least one of PBS, EDTA, and BSA wherein the volume ratio ofbuoyant particles to target constituent is 3:1, with a dwell time of 20minutes. However, any suitable combination of substrate, targetconstituent, solvent, and/or additional binding agents and respectivedwell times can be used in any other suitable manner to achieve anyvalue of complexification binding efficacy.

After combination of substrates and target constituents in Block S110,the final concentrations of substrate particles, final concentration oftarget constituents, and the total sample volume in the process chambercan vary depending on the type of target constituent (e.g., red bloodcell, white blood cell, T cells, B Cells, etc.), the percentagecomposition of the target constituent in the target sample (e.g., TCells can make up to approximately 50% of cells in a human peripheralblood mononuclear cell sample), the total number of target constituents,the rate of complexification, the buffer used in the target samplesolution containing the target constituents, and/or any other suitablefactor. For example, an initial target sample concentration of 40,000cells per microliter fluidic volume can be processed (e.g., captured andseparated) by obtaining (e.g., by dilution) a final total sampleconcentration, wherein the final substrate concentration ranges from7,400 to 55,000 substrate particles per microliter and the final targetconstituent concentration ranges from 18,000 to 37,000 cells permicroliter. In another example, an initial target sample concentrationof 200,000 cells per microliter fluidic volume can be processed byobtaining a final total sample concentration wherein the final substrateconcentration ranges from 25,000 to 90,000 substrate particles permicroliter and the final target constituent concentration ranges from29,000 to 145,000 cells per microliter. However, the finalconcentrations of each of the substrate particles, the targetconstituents, target-bound complexes, and/or any other component of thetotal sample can be otherwise configured.

In Block S110, combination of the substrate volume with the targetsample volume is preferably achieved using trituration methods; howevercombination can be achieved by end over end rotation, shaking, and/orany other suitable method to thoroughly combine the substrate volumewith the target sample volume for complexification. In a variation,trituration is used to combine the substrate volume with the targetsample volume, using a pipette (e.g., syringe, pump, hydraulic device,etc.) configured to transfer (e.g., add, remove) up to 50-100% of thefluidic volume from the process chamber up to 60 repetitions. However,combining the substrate volume and the target sample volume can beachieved in any other suitable manner.

Furthermore, combination in Block S110 is preferably performed in amanner that prevents damage to (e.g., due to shear forces, due to otherforces) or destruction of substrates of the volume of substrates and/orelements of the target constituent. In one variation, the target samplevolume can include a buffer solution configured to provide abiocompatible (e.g., non-toxic, non-hazardous) environment for targetconstituents. In a specific example, buffer solution includes at leastone of PBS, BSA, and/or EDTA. In another variation, the target samplevolume can include a buffer solution configured to maintain thestability of the substrate particles and includes at least a combinationof PBS, EDTA, and NaCl. However, the buffer solution can include anyother suitable reagent, growth factor, chemical compounds, solvent,and/or be of any suitable pH, temperature, or other characteristic tosupport the viability of substrate particles (e.g., minimize particleaggregation, improve longterm storage, etc.) and/or target constituents.Additionally, combining in Block S110 can be performed in a manner thatprevents foaming of sample volumes, which can impede separation of thetarget constituent from the sample in subsequent blocks of the methodaccording to buoyancy-based approaches. For instance, combining in BlockS110 can be performed with a characteristic velocity (e.g., linearvelocity, angular velocity) and/or below a desired level of accelerationof the process chamber in order to prevent foaming of the sample.However, combining in Block S110 can additionally or alternatively beperformed in a manner that facilitates lysing of sample components(e.g., non-target sample components, target sample components wherelysing releases the target constituent for binding to the volume ofsubstrates). For instance, mixing to lyse untargeted sample componentscan facilitate subsequent separation of the untargeted sample componentsfrom the target constituents of the sample. Additionally oralternatively, lysing portions of the sample can facilitate release ofthe target constituent into solution for binding to the volume ofsubstrates. However, combining in Block S110 can alternatively beperformed in any other suitable manner.

In variations for multiplex separation and/or processing of a set oftarget constituents of the sample, the volume of substrates used inBlock S110 can additionally or alternatively be configured toselectively bind to one of a set of target constituents of the sample.In one variation, the volume of substrates can comprise a first subsetof substrates having a first density and processed with a first moietyconfigured to target a first target constituent, a second subset ofsubstrates having a second density and processed with a second moietyconfigured to target a second target constituent, and any other suitablenumber of subsets of substrates having distinguishable densities andprocessed with specific moieties for targeting any other suitable numberof target constituents of the sample. In one such example, the volume ofsubstrates can include a first subset of substrates having a firstdensity and processed with a moiety for targeting CD133+ expressingcells, a second subset of substrates having a second density (differentform the first density) and processed with a moiety for targeting CD15+expressing cells, and a third subset of substrates having a thirddensity (different from the first and the second densities) andprocessed with moieties for targeting CD133+ and CD15+ expressing cells.In the example, specific subsets of the set of substrates can thusfacilitate selective separation of cells expressing different biomarkersfor further analysis. However, alternative variations of the volume ofsubstrates can include subsets of substrates having any other features(e.g., physical feature, chemical feature, etc.) and configured to bindto different target constituents of the sample, in order to facilitateselective separation of different target constituents from the sample.Furthermore, in some variations, at least a subset of the volume ofsubstrates can be configured to bind to and facilitate separation of anuntargeted (e.g., waste, debris, etc.) portion of the sample.

In Block S110, combining can be supported by or supplemented withprovision of environmental conditions and/or additional processreagents, to support subsequent processing steps and/or analysis of thetarget constituent. In one variation, mixing can be supplemented withmodulating a temperature within the process chamber (e.g., to facilitatelysis or binding of the target constituent to the volume of substrates,to thermocycle the sample, etc.). Additionally or alternatively, mixingcan include providing a lysing reagent (e.g., lysing solution, beadbeating solution, etc.) within the process chamber, along with thesample and the volume of substrates, in order to facilitate lysis ofundesired constituents of the sample and/or lysis of portions of thesample to release the target constituent for binding to the volume ofsubstrates. Additionally or alternatively, mixing can include providinga fixing reagent (e.g., a cross-linking reagent) configured to fixportions of the sample. Additionally or alternatively, mixing caninclude providing a pH modulating reagent within the process chamber,and/or any other suitable reagent configured to provide a desiredenvironment within the process chamber. However, Block S110 can beperformed using any other suitable combination of reagents and/oradjustments to temperature, pH, or composition.

1.2 Method—Washing and Separation

Block S120 recites: within the process chamber, physically separatingthe population of target-bound complexes from the sample based uponinteraction between the volume of substrates and an applied force.Similar to Block S110, Block S120 is preferably performed within anembodiment, variation, or example of the process chamber described inSection 2 below; however, Block S120 can additionally or alternativelybe performed using any other suitable process chamber. Furthermore,while Blocks S110 and S120 are preferably implemented within the sameprocess chamber, Blocks S110 and S120 can alternatively be implementedusing separate process chambers.

Block S120 functions to enable distinction of the population oftarget-bound complexes from other portions of the sample. In Block S120,physical separation can include promoting passive buoyant separation,within the process chamber, whereby the population of target-boundcomplexes experience buoyant forces due to effects of gravitationalforce on the volume of substrates having the first density, differentfrom the densities of other untargeted constituents of the sample. Thus,a gravitational force can allow the population of target-bound complexesto move opposite a direction of the gravitational force for collectionin Block S130. Additionally or alternatively, in Block S120, physicalseparation can include promoting active buoyant separation, within theprocess chamber, whereby the population of target-bound complexesexperience buoyant forces due to effects of centrifugal force about anaxis of rotation of the process chamber (e.g., as applied by acentrifuge interacting with the process chamber). Implementing activebuoyant separation can further increase a separation effect over that ofpassive buoyant separation alone. Embodiments, variations, and/orexamples of Block S110 are further described in U.S. application Ser.No. 14/969,446, filed 15 Dec. 2015.

In relation to separation in Block S120, embodiments of the method 100can additionally or alternatively include Block S122, as shown in FIG.1B, which recites: within the process chamber, washing the population oftarget-bound complexes. Block S122 functions to facilitate purificationand/or enrichment of the population of target-bound complexes within theprocess chamber, prior to extraction of the population of target-boundcomplexes from the process chamber in subsequent blocks of the method100. As such, Block S122 can facilitate removal of the non-targetconstituents of the sample in promoting a higher degree of efficiency inextraction of the target constituent from the sample.

Preferably, as shown in FIGS. 1B and 1D, Block S122 includes bringingthe population of target-bound complexes to a desired region of theprocess chamber with an applied force; adjusting an orientation of theprocess chamber, thereby promoting the population of target-boundcomplexes to adhere to a wall of the process chamber; removingnon-target portions of the sample from the process chamber; andresuspending the population of target-bound complexes within the processchamber with a suspension volume of fluid having a sufficiently highdensity. Washing in Block S122 can be performed any suitable number oftimes prior to extraction of the population of target-bound complexes insubsequent blocks of the method 100.

In Block S122, bringing the population of target-bound complexes to adesired region of the process chamber with an applied force can includeallowing the population of target-bound complexes to undergo passivebuoyant separation, within the process chamber, whereby the populationof target-bound complexes experience buoyant forces due to effects ofgravitational force on the volume of substrates having the firstdensity, different from the densities of other untargeted constituentsof the sample. Thus, a gravitational force can allow the population oftarget-bound complexes to move opposite a direction of the gravitationalforce in support of the washing operation of Block S122. Additionally oralternatively, in Block S122, physical separation can include promotingactive buoyant separation, within the process chamber, whereby thepopulation of target-bound complexes experience buoyant forces due toeffects of centrifugal force about an axis of rotation of the processchamber (e.g., as applied by a centrifuge interacting with the processchamber). Additionally or alternatively, physical separation can beimplemented in any other suitable manner.

In Block S122, adjusting an orientation of the process chamber,functions to allow the population of target-bound complexes to adhere toa wall of the process chamber. In more detail, tilting of the processchamber can allow the population of target-bound complexes to aggregateat an anti-dependent rim of a fluid meniscus formed within the processchamber (e.g., an upper rim of a fluid meniscus in the orientation shownin FIG. 1D), after which removal of non-target portions of the samplefrom the process chamber can be performed (e.g., by pipetting, etc.).Thus, with gentle aspiration, non-target components of the sample, aswell as a bulk of the sample fluid, can be removed from the processchamber while a substantially purified volume of the population oftarget-bound complexes is retained at the wall of the process chamber.Finally, the population of target-bound complexes can be washed from thewall of the process chamber and resuspended within a volume of fluid,where the volume of fluid is similar to, greater than, or substantiallysmaller than the original volume of fluid in the sample.

Additionally or alternatively, to enhance retention of the population oftarget-bound complexes at the process chamber during the washing processof Block S122, interior surfaces of the process chamber, proximal thecollection region(s), can be patterned (e.g., roughened, texturized byetching, textured by molding, tapped/threaded, etc.) to enhanceretention of the population of target-bound complexes at desired regionsof the process chamber, as described in Section 2 below. Additionally oralternatively, retention of the target-bound complexes at regions of theprocess chamber can be enhanced by utilizing a recessed region or otherregion of the process chamber defining a separated interior volume ofthe process chamber, configured to retain the population of target-boundcomplexes during washing. Additionally or alternatively, washing inBlock S122 can be enhanced by including a high molecular weight polymerwith a washing solution, thereby promoting the generation of colloidalforces to reversibly enhance aggregation of the population oftarget-bound complexes within the fluid meniscus of the process chamber.

Block S122 can be performed manually (e.g., by an technician or otherentity), or can alternatively be performed in an automated manner (e.g.,with an automated system for adjusting the orientation of the processchamber, and with a fluid handling system for aspirating the non-targetcomponents of the sample/washing the target components of the sample).Block S122 can, however, be performed in any other suitable manner.

1.3 Method—Aggregation at a Collection Region

Block S130 recites: aggregating the population of target-bound complexesat a collection region of the process chamber, which functions toaggregate the population of target-bound complexes to at least onedesired region of the process chamber, in order to facilitate extractionof the target constituent from the sample in an efficient manner.Interactions (e.g., hydrophobic interactions, hydrophilic interactions,neutral interactions) between fluid of the sample and the processchamber, and/or the method of separation (e.g., passive buoyantseparation, active buoyant separation, compound density gradient, etc.)can affect the location(s) of the collection region(s) of the processchamber at which the population of target-bound complexes reside.Embodiments, variations, and/or examples of Block S110 are furtherdescribed in U.S. application Ser. No. 14/969,446, filed 15 Dec. 2015.

In some variations, implementation of passive buoyant separation inBlock S120 can provide one or more collection region profiles, dependingupon interaction of the process chamber with fluid of the sample. Asshown in FIG. 2A, in variations of Block S120 that include a hydrophilicinteraction between fluid of the sample and the process chamber, aconcave meniscus formed at the fluid surface can provide a collectionregion at the perimeter of the fluid surface (e.g., a circular annularcollection region for a cylindrical process chamber, a rectangularannular collection region for a rectangular prismatic process chamber,etc.) where clearing or an otherwise lower density population oftarget-bound complexes within the center of the meniscus can be used toextract bulk sample fluid and leave the population of target-boundcomplexes within the process chamber. However, as shown in FIG. 2B, themeniscus can also be convex, wherein a higher density population oftarget-bound complexes occurs within the center of the fluid surface,allowing extraction of the target-bound complexes away from thecollection region at the center of the process chamber. As shown in FIG.2C, in variations of Block S120 that include a neutral (e.g.,non-hydrophobic, non-hydrophilic) interaction between fluid of thesample and the process chamber, a substantially planar surface formed atthe fluid surface can provide a collection region at the fluid surface.Examples of process chambers contributing to a concave or a convex fluidmeniscus associated with a sample volume can include one or more of: amicrowell plate, a microfabricated array, any other suitable well plate,any other suitable fabricated array, and any other suitable processchamber. Furthermore, as shown in FIG. 2D, the use of various reagents(e.g., water, PBS, RPMI, with or without protein additives) can be usedto influence the shape of the fluid meniscus. In more detail, the shapeof the meniscus can be influenced by the interaction of the well plate(e.g., material composition, surface treatments, sidewall features,morphological characteristics) with the sample fluid within the well,and additionally and/or alternatively can be modified by combiningvarious buffers (e.g., containing different solutions, salts,surfactants, etc.) within the total sample volume (e.g., to influencesurface tension of the sample fluid). However, any suitable reagentand/or solvent can be used to perform Block S130 and/or any otherportion of method 100.

In any of the above variations, morphology and/or concentration of thecollection region(s) can be enhanced by providing process chambermorphologies that affect the morphology(ies) of the collectionregion(s). For instance, in the examples shown in FIGS. 3A-3D, a conicalprocess chamber morphology (e.g., linear conical, curved conical, etc.)with the vertex oriented at a superior portion of the process chambercan concentrate the collection region to a smaller region in comparisonto a process chamber having a substantially constant or wideningcross-section (i.e., in an inferior to superior direction) along alongitudinal axis of the process chamber. In one example, as shown inFIG. 4, a frustoconical process chamber having a vertex oriented at asuperior portion of the process chamber can concentrate the collectionregion, and an opening at the vertex of the frustoconical processchamber can enhance separation and facilitate collection of thepopulation of target-bound complexes at the opening. Furthermore,overfilling of the frustoconical process chamber with the sample canprovide a convex fluid surface at the opening, in the example, thateffectively concentrates the population of target-bound complexes to asingle location for extraction in Block S140. However, any othersuitable morphology of process chamber, another example of which isshown in FIG. 5, can be used to produce any shape of collection regionand/or any number of collection regions within a process chamber.Furthermore, interior surface features of the process chamber canenhance collection in Block S130, as described in Section 2. In oneexample, a process chamber that has one or more recessed regions (e.g.,scoring lines) at given positions (e.g., heights) along its interiorsurface (e.g., sidewall) can facilitate collection of the population oftarget-bound complexes within the recessed region(s) for laterextraction in Block S140. In this example, the recessed regions canfacilitate repeated separation and washing of the population oftarget-bound complexes within the process chamber. In a specificexample, a 1.5 mL centrifuge tube used as the process chamber can betapped, wherein a set of internal threads along the sidewall of thecentrifuge tube can be formed using an M9-sized tap or any othersuitable tool (e.g., spiral flute tap, spiral point tap, pin, plug,etc.). The modified (e.g., threaded) sidewalls of the centrifuge tubecan then be used to efficiently capture target-bound complexes aroundthe entire circumference and upper section of the tube (e.g., due tosurface tension between the sample fluid and the set of internal threadsof the sidewall), However, the process chamber can be otherwise modifiedand/or configured as described in Section 2.

In a specific example, aggregating the population of target-boundcomplexes can comprise aggregating the population of target-boundcomplexes at a superior portion of a process chamber (e.g., at aliquid-air interface of a sample volume within the process chamber),based upon passive buoyant separation. However, variations of theexample can comprise any other suitable region for aggregation.

In variations of Block S120 implementing an active buoyant separationprocess, an axis or point about which the process chamber rotatesgoverns the location of the collection region in Block S130. Inparticular, the population of target-bound complexes and/or substratesof the volume of substrates will migrate in a direction opposite that ofa centrifugal or gravitational force. In one variation, wherein theprocess chamber rotates about a radial axis, as shown in FIG. 6A, lowerdensity particles (e.g., particles of the population of target-boundcomplexes, substrates of the volume of substrates) in the sample movetoward the radial axis and higher density particles move away from theradial axis. As such, the collection region in this variation ispositioned at the portion(s) of the process chamber that were closest tothe radial axis during rotation of the process chamber about the radialaxis. In another variation, wherein the process chamber rotates about alongitudinal axis of the process chamber, as shown in FIG. 6B, lowerdensity particles (e.g., particles of the population of target-boundcomplexes, substrates of the volume of substrates) in the sample movetoward the longitudinal axis and higher density particles move away fromthe longitudinal axis. As such, the collection region in this variationis positioned at the portion(s) of the process chamber that were closestto the longitudinal axis during rotation of the process chamber aboutthe longitudinal axis. Furthermore, in variations wherein rotationoccurs about a longitudinal axis of the process chamber, a rate at whichlower density particles float can be increased due to enhancement ofbuoyant effects resulting from increased proximity between multiple lowdensity particles.

In more detail, and in relation to either passive or active buoyantseparation, Block S130 can include enhancing a speed at whichaggregation occurs by promoting a reduction in proximity betweencomplexes of the population of target-bound complexes. As such, themethod 100 can include Block S135, as shown in FIG. 1B, which recites:transitioning the process chamber into a first orientation that promotesan increase in a complex-to-complex proximity metric for the set oftarget-bound complexes. Block S135 can function to increase an effectiveradius, r_(eff), and therefore, to increase an effective volume ofaggregates of the population of target-bound complexes, therebyincreasing the effective buoyant forces on aggregates of the populationof target-bound complexes. As shown in FIG. 1E, as the number of buoyantparticles within an aggregate increases, the net buoyant force, andtherefore the terminal velocity of the aggregate increases, therebyenhancing a speed at which aggregation occurs.

In one variation, as shown in FIGS. 1G-1I, Block S135 can includeimplementing a process chamber that has a high aspect ratio (e.g.,rectangular prism), wherein the process chamber is transitioned into afirst orientation that promotes an increase in a complex-to-complexproximity metric for the set of target-bound complexes (e.g., theprocess chamber is transitioned onto its side). In this variation, thefirst orientation can be associated with an orientation wherein a shortaxis of the process chamber is substantially aligned with a direction ofgravity. Maintenance of this orientation for a duration of time thusallows buoyant particles to move through the shortest possible distancein forming aggregates. Then, Block S135 can include transitioning theprocess chamber from the first orientation to a second orientation thatdrives the set of target-bound complexes toward a collection region ofthe process chamber. The second orientation can be associated with anorientation wherein a long axis of the process chamber is substantiallyaligned with a direction of gravity. This orientation thus allows theformed aggregates to aggregate toward the collection region of theprocess chamber in a manner that is more rapid than that of individualbuoyant particles, due to a combination of hydrodynamic drafting amongbuoyant particles and the formation of transient particle aggregateswith a much greater effective volume.

In a specific example, as shown in FIG. 1J and FIGS. 1K-1M, arectangular process chamber 500 can be used to improve the speed ofseparation of target-bound complexes from a large volume of solution(e.g., 25 mL, 100 mL, 1000 mL, etc.). The rectangular process chamber500, further described in Section 2, includes a rectangular prismaticmorphology having a high aspect ratio (e.g., vertical height is greaterthan the width) with walls configured at an angular offset from vertical(e.g., 1°, 3°, 5°, 15°, etc.). As shown in FIGS. 1K-1M, after the sampleis added to the rectangular process chamber, separation can occur inthree steps, wherein each step can include turning the process chamberonto one of three exterior surfaces with a respective dwell time (e.g.,1-20 minutes duration on each side) for each step, thus concentratingthe target-bound complexes against an opposing interior sidewall, edgeof a sidewall, and/or any other suitable surface of the process chamber.In a first step (FIG. 1K), the rectangular process chamber 500 can reston its broad surface 510, allowing the target-bound complexes to rise tothe opposing first sidewall 510′. In a second step (FIG. 1L), therectangular process chamber 500 is rotated about its longitudinal axisby 90° to rest on its side surface 530, thus allowing the target-boundcomplexes to aggregate against the sidewall edge 535 between the firstsidewall 510′ and a second sidewall 530′ opposing the side surface 530.In a third step (FIG. 1M), the rectangular process chamber 500 isrotated about its horizontal axis by 90°, bringing the rectangularprocess chamber 500 into its upright position, and enabling increasedvelocity of the target-bound complexes to the collection region 570(e.g., upper region of the process chamber 500), due to the improvedcomplex-to-complex proximity metric of buoyant particles at the sidewalledge in the second step. However, adjusting the orientation of theprocess chamber can be performed in any other suitable manner, with anynumber of steps, and/or with any other rate of transition betweenorientations (e.g., fixed steps, constant rotation, rapid movements,multiple axes, etc.) in order to efficiently separate the target-boundcomplexes from solution.

In other variations, Block S135 can additionally or alternativelyinclude actively forming aggregates of subsets of the population oftarget-bound complexes based upon one or more of: centrifugation of theprocess chamber about any suitable axis; binding of multiple buoyantsubstrate particles to each unit of the target constituent of thesample; use of high-molecular weight polymer solutions that promoteaggregation of the population of target-bound complexes in a reversiblemanner; and any other suitable means to promote aggregation. In relationto binding of multiple buoyant substrate particles to each unit of thetarget constituent of the sample, binding can be tuned such that risingof an aggregate within the process chamber only occurs when a desirednumber of buoyant components (e.g., 2 or more buoyant substrates) arecoupled to a target. However, variations of Block S135 can additionallyor alternatively be implemented in any other suitable manner.Furthermore, in any of the above variations of separation, separationcan be used to indicate presence of the target constituent (e.g., usingnatural coloring of the target constituent, using stains, etc.), wherebymonitoring of completion of a binding process based on completeness of aband of separation can additionally or alternatively be implemented.

1.4 Method—Extraction

Block S140 recites: extracting the population of target-bound complexesfrom the process chamber, which functions to remove the population oftarget-bound complexes from other portions of the sample, or to removeportions of the sample from the population of target-bound complexes,thereby generating an enriched volume comprising the population oftarget-bound complexes. Block S140 preferably includes transmitting atarget constituent extractor into the process chamber, concentrating thepopulation of target-bound complexes at a region of the targetconstituent extractor, and delivering the population of target-boundcomplexes from the target constituent extractor for downstreamprocessing. Embodiments, variations, and/or examples of Block S110 arefurther described in U.S. application Ser. No. 14/969,446, filed 15 Dec.2015.

In one variation, as described in more detail in Section 2 below, thetarget constituent extractor can include opposing frustoconical surfaces(FIGS. 15A-15D) configured to facilitate concentration and extraction ofthe population of target-bound complexes from the process chamber.However, the target constituent extractor can alternatively include asingle frustoconical surface facing the surface of the sample fluid,and/or be otherwise configured as described in Section 2. In one exampleof this variation, the target constituent extractor can include athreaded region that complements threads of the process chamber (e.g., a50 mL tube), such that transmitting the target constituent extractorincludes rotating the target constituent extractor relative to theprocess chamber in order to engage their complementary threads. Inanother example of this variation, the target constituent extractor mayomit threads, transmitting the target constituent extractor into theprocess chamber can include translating the target constituent extractorconcentrically into an opening of the process chamber. Transmitting thetarget constituent extractor into the process chamber can, however, beimplemented in any other suitable manner. Once inserted into the processchamber, the target constituent extractor can be removably secured;however, the target constituent extractor and/or portions thereof canalternatively be fixed in place (e.g., permanently secured), orotherwise configured (e.g., FIGS. 21A-21B).

In relation to the variation described above, concentrating thepopulation of target-bound complexes at a region of the targetconstituent extractor can include concentrating the set of target-boundcomplexes at a region of a frustoconical surface of the targetconstituent extractor, wherein the target constituent extractor has aninverted frustoconical surface opposing and coupled to the frustoconicalsurface by a channel. Then, to facilitate delivery of the population oftarget-bound complexes from the target constituent extractor, a fluidlevel within the inverted frustoconical surface can be adjusted (e.g.,by exertion of an applied force, change in pressure, process chambermovement, solution agitation, etc.), in order to bring the population oftarget-bound complexes into the inverted frustoconical surface forextraction. In one example, the fluid level can be adjusted uponreceiving an additional fluid volume into the channel of the targetconstituent extractor connecting the two frustoconical surfaces, therebydelivering the population of target-bound complexes from thefrustoconical surface and into the inverted frustoconical surface by wayof the channel. In another example, the fluid level can be adjusted bytranslating at least a portion of the target constituent extractordeeper into the process chamber, thereby applying a change in pressureconfigured to alter a volume of air and or/solution within the processchamber to deliver the population of target-bound complexes from thefrustoconical surface and into the inverted frustoconical surface by wayof the channel. In another example, wherein the process chamber isdeformable (e.g., squeezable), the fluid level can be adjusted upondeformation of the process chamber (e.g., squeezing of the processchamber), thereby delivering the population of target-bound complexesfrom the frustoconical surface and into the inverted frustoconicalsurface by way of the channel. However, adjusting the fluid level canadditionally or alternatively be implemented in any other suitablemanner.

Finally, in Block S140, delivering the population of target-boundcomplexes from the target constituent extractor for downstreamprocessing can include delivering the population of target-boundcomplexes into a pipette for extraction of the population oftarget-bound complexes from the target-constituent extractor.Additionally or alternatively, in a variation of the target constituentextractor including a separate extraction component having a secondinverted frustoconical surface opposing the inverted frustoconicalsurface, as shown in FIG. 15A, delivering the population of target-boundcomplexes can include delivering the population of target-boundcomplexes into the second inverted frustoconical surface of the separateextraction component (e.g., without involvement of a pipette).Variations of extraction of the population of target-bound complexescan, however, be implemented in any other suitable manner.

In one embodiment of the system using the variation of the processchamber shown in FIG. 1J and the variation of the extraction apparatusshown in FIGS. 21A-21B, the target constituent extractor can include acontinuous fluidic pathway from the process chamber to an upper chamber(e.g., a separate collection tube, secondary container), wherein theupper chamber can be sealed (e.g., to prevent airflow) with a separatorlid, such that the target-bound complexes can be maintained in isolationwithin the upper chamber when the target constituent extractor iscompletely removed from the process chamber. In one example, as shown inFIG. 21B, a portion of the target constituent extractor (e.g., afrustoconical surface facing the surface of the sample fluid) can beconfigured as a collection plunger that operates in coordination with avalve slit coupled to a vent path, which regulate the air pressurewithin the system. When the target extraction apparatus is coupled tothe process chamber, the collection plunger can be arranged in a firstconfiguration (e.g., lowered configuration) to enable target-boundcomplexes to travel into the upper chamber, wherein the valve slit isclosed and air is unable to pass through the vent path. Once transfer iscomplete, the upper chamber can be secured with the separator lid andremoved from the sample containing chamber, thus opening the vent pathto allow air to pass through the valve slit, maintaining air pressurewithin either of the upper chamber and the process chamber, such thatthe fluid in the upper chamber stays in the upper chamber. In onevariation, the radius of the opening at 363′ is configured to be lessthan the radius of the opening of 364,′ which functions to allowtarget-bound complexes to pass from the process chamber to the upperchamber without being caught (e.g., stuck, unflowing, immobile) at theintersection of the two chambers. After target-bound complexes have beenrecovered in the upper chamber, the extraction apparatus (and theenriched sample captured in the upper chamber) can be removed from theprocess chamber, whereby the upper chamber can be coupled to avertically aligned plug inserted into the upper chamber, and/or anyother suitable sealing mechanism that fluidly seals the lower portion ofthe upper chamber, thus allowing the separator lid to be removed fromthe upper chamber without losing the enriched sample fluid contained inthe upper chamber (e.g., due to pressure management within the processchamber and/or upper chamber). However, the extraction apparatus can beotherwise configured.

In removing fluid (e.g., fluid containing the population of target-boundcomplexes, fluid excluding the population of target-bound complexes)using the target constituent extractor, removal can include active fluidremoval (e.g., by pipetting, by capillary action, etc.). Additionally oralternatively, fluid removal can include surface contact methods,whereby an extraction element (e.g., target constituent extractordescribed below) makes surface contact with fluid of the sample in orderto provide extraction. In extraction, fluidic transfer can occurthrough 1) wetting/capillary forces when an extraction element (e.g.,extraction vessel, extraction membrane) makes contact with thecollection region containing the population of target-bound complexesand/or 2) further climbing of buoyant elements (e.g., the population oftarget-bound complexes, substrates of the volume of substrates) withinan extraction element. Extraction elements can include one or more of:solid substrates with or without a patterned surface (e.g., a glasssubstrate with a Teflon pattern to define an active extraction region);capillary structures (e.g., an element with a tubular geometry); annularelements (e.g., a ring, a circular annular element, a rectangularannular element, etc.); and any other suitable element configured topromote fluid transfer.

In another example of Block S140 wherein a hydrophilic interactionoccurs between fluid of the sample and the process chamber, a regionsubstantially void of the population target-bound complexes can becreated proximal a central longitudinal axis of the process chamber, asshown in FIG. 8. In this example, extraction can comprise removal offluid from the region that is void of the population of target-boundcomplexes, thus concentrating the population of target-bound complexeswithin the process chamber. To enhance retention of the population oftarget-bound complexes at the process chamber and/or the targetconstituent extractor, interior surfaces of the process chamber and/orportions of the target constituent extractor, proximal the collectionregion(s), can be patterned (e.g., roughened, texturized by etching,textured by molding, tapped/threaded, etc.) to enhance retention of thepopulation of target-bound complexes at the process chamber, as furtherdescribed in Section 2. Furthermore, in this example, single-instance orrepeated washing (e.g., with a wash buffer) and extraction of fluid fromthe region that is void of the population of target-bound complexes canfurther enrich the population of target-bound complexes within theprocess chamber, as described in relation to Block S122 above.Furthermore, automation of the fluid extraction process (e.g., with anautomated liquid handling system) can further remove burden for atechnician or other entity associated with the example of Block S140.

While several extraction elements and methods of use are described, anyother suitable combination of the above described extraction elements,and/or any other suitable extraction element(s) can facilitateextraction of the population of target-bound complexes in Block S140.Furthermore, while the above Blocks are described in distinction fromeach other, any one or more of the embodiments, variations, and/orexamples of Blocks S110-S140 can be performed substantiallysimultaneously with each other, in order to provide an efficient sampleprocessing mechanism. As such, in some variations, a process chamberused in the method 100 can be module and comprise a first portion forcollection and a second portion for extraction of the population oftarget-bound complexes, such that collection and extraction can occursimultaneously within a single process chamber. In some variations, theprocess chamber can further be configured to facilitate simultaneouscombination of the volume of substrates with the target constituent, andcollection of the population of target-bound complexes, bysimultaneously mixing and driving the population of target-boundcomplexes toward the collection region(s). Simultaneous performance ofmultiple blocks of the method 100 can, however, be implemented in anyother suitable manner.

1.5 Method—Downstream Processing

Block S150 recites: processing the target constituent from thepopulation of target-bound complexes for further analysis. Block S150functions to provide a means for subsequent processing of the targetconstituent of the sample for a downstream application. Block S150 cancomprise one or more of: Block S160, which recites resuspending thepopulation of target-bound complexes for at least one of analysis andstorage; and Block S170, which recites generating an analysis of thetarget constituent upon processing of the population of target-boundcomplexes. Embodiments, variations, and/or examples of Block S150, BlockS160, and Block S170 are further described in U.S. application Ser. No.14/969,446, filed 15 Dec. 2015.

In some variations the method 100 can include carrying out surfacechemistry and/or other modifications to substrates of the volume ofsubstrates, in order to enhance processing and separation of the targetconstituent from the sample. Such modifications can be implemented usingprocess chamber morphologies, described further in Section 2 below,which allow multistep modifications to be performed on substratesurfaces without requiring direct handling of the substrates. As such,handling of buoyant substrates can be performed in a manner that reducesproduct loss and handling of potentially harmful reagents. In one suchvariation, a process chamber having a dip tube that provides access tofluid within the process chamber, below an active collection region atwhich buoyant substrates aggregate, can enable fluid transfer in theprocess chamber substantially without disturbance of the buoyantsubstrates. In examples, the dip tube can be incorporated into a lid ofthe process chamber, as shown in FIG. 12A, incorporated into a wall ofthe process chamber, as shown in FIG. 12B, be a modular component thatinterfaces with the process chamber (e.g., by adhering to a wall of theprocess chamber with a membrane), and/or cooperate with the processchamber in any other suitable manner.

1.6 Method—Additional Examples

In one example workflow for modification of a buoyant substrate, asshown in FIG. 10, the method can include introducing buoyant substrates(e.g., beads in powder form, beads suspended in liquid) into the processchamber (A); adding a first reaction solvent and associated reactantsinto the process chamber, by way of a dip tube coupled to the processchamber (B); incubating the buoyant substrates with the first reactionsolvent to achieve chemical modification (C); centrifuging the processchamber (D) to achieve separation of the modified buoyant substrates(E); removing excess portions of the first reaction solvent andreactants from the process chamber, by way of the dip tube (F);preparing the process chamber for additional modification solvents andreactions (G); and repeating steps (A)-(G) as necessary (H).

In a specific example, method 100 can be used to enrich (e.g., separate)murine B cells from splenocytes derived from mice by using the substrateparticles to bind to non-target constituents (e.g., non-murine B cells).However, the method 100 can alternatively be used to isolate any othersuitable cell type, and the target-bound complexes can include eithercells of interest (e.g., to be used for further analysis), cells ofnon-interest (e.g., to be discarded), and/or any other suitablecomponent of the sample volume. In this example, method 100 includes: ata process chamber, generating a target sample volume including mousesplenocytes at an initial target concentration of 2×10⁶ cells in 100 μLvolume of separation buffer; combining the target sample volume with asubstrate volume for a total sample volume of 200 μL, wherein thesubstrate volume includes an initial substrate concentration of 4million streptavidin-coated glass microbubbles (e.g., 2 substrateparticles per splenocyte); for each process chamber, triturating thesample volume with 60 strokes (1/second for 1 minute); centrifuging theprocess chamber; and, upon separation of an enriched cell pellet fromthe microbubbles, aspirating the volume of microbubbles from the uppersurface of the sample volume, leaving an enriched murine B cell pelletintact at the base of the tube that can be used for additionalprocessing steps (e.g., for further analysis).

The method 100, can, however, include any other suitable Blocks or Stepsfor separating a target constituent from a sample based upon buoyancyand/or any other separation mechanism, and generating an analysis basedupon processing of the target constituent. Furthermore, as a personskilled in the art will recognize from the previous detailed descriptionand from the figures, modifications and changes can be made to themethod 100 without departing from the scope of the method 100.

2. System 2.1 System—Process Chamber

As shown in FIG. 11, an embodiment of a process chamber 200 for buoyantseparation of a target constituent of a sample comprises: afrustoconical surface 210 defining a base region 220 having a firstwidth and a collection region 230 having a second width, narrower thanthe first width, in opposition to the base region, wherein thefrustoconical surface defines a volume 240 configured to receive thesample having the target constituent. In variations, the frustoconicalsurface can have a straight profile in elevation, as shown in FIGS. 3Aand 3D. Alternatively, the frustoconical surface can have a curvedprofile or any other suitable profile in elevation, as shown in FIGS.3B-3C. However, embodiments of the process chamber can be otherwiseconfigured.

The volume 240 defined within the process chamber 200 preferably has alow height-to-width ratio, in order to facilitate rapid separation ofthe target constituent from the sample (e.g., by providing a smallertraveling distance for a given volume). However, the volume 240 definedwithin the process chamber 200 can alternatively have a highheight-to-width ratio (e.g., as a slender volume, rectangle, cylinder),or any other suitable height-to-width ratio. However, the processchamber can be of any other suitable geometry.

The process chamber is preferably fabricated using glass or plasticmaterials, such as polystyrene, polypropylene, polycarbonate,cycloolefin, and/or glass. Furthermore, any surface (exterior and/orinterior) of the process chamber can be optionally treated with asurface coating (e.g., to influence surface properties, adhesionproperties, optical properties, etc.). However, the process chamber canbe configured in any other suitable manner.

The process chamber 200 is preferably used to perform any combination ofBlock S110, Block S120, and/or Block S130, as described in Section 1,but can be used to perform any other suitable portion of the method 100.Embodiments, variations, and/or examples of the process chamber arefurther described in U.S. application Ser. No. 14/969,446, filed 15 Dec.2015.

In variations, the process chamber 200 can be adapted from acommercially available (e.g., pre-manufactured) chamber, dish, vial,tube, well, microwell plate, or any other suitable container forhandling volumes of fluids (e.g., as shown in FIG. 1D), but canalternatively be left unaltered. In examples, the process chamber is a1.5 mL centrifuge tube, but can also be a 15 mL tube, 50 mL tube, or anyother vessel configured to contain any other suitable volume. In otherexamples, the process chamber can be one of a plurality of processchambers within a multi-well plate (e.g., 96 well, 385 wells, 1536wells, etc.) (e.g., as shown in FIG. 2D), enabling integration withautomated sample handling systems and multiplexed sample processing inparallel (e.g., when the plates are stacked).

In an alternative variation of the process chamber, as shown in FIG. 1J,the process chamber can be a rectangular prism, or any other suitablegeometry with a high aspect ratio, and configured to hold a large samplevolume (e.g., 100 mL as opposed to 1.5 mL). To enhance separationtowards the walls and then flotation to the top surface, each of thewalls of the rectangular process chamber 500 are preferably configuredat a 3° offset from vertical, but can be any other suitable angle (e.g.,1°, 5°, 10°, 15°, etc.). As shown in FIG. 1J, a specific example of therectangular process chamber 500 has a broad face 510, a side face 530,and a bottom face 550, with a collection region 570 at the upper regionof the process chamber 500. However, the process chamber 500 can includeany number of faces with any suitable dimensions. A variation of themethod utilizing the rectangular process chamber is described in Section1 (Block S135). However, the rectangular process chamber can be in anyother suitable manner to effectively promote an increase incomplex-to-complex proximity metric for the set of target-boundcomplexes. Furthermore, the rectangular process chamber 500 canadditionally include a solution addition unit 590 to allow for easysolution pouring into the rectangular process chamber. The solutionaddition unit 590 is preferably located at a corner of the upper surfaceof the process chamber opposing the bottom face 550, and proximal to thecollection region 570, but can alternatively be positioned at any othersuitable location to permit facile loading and/or extraction of fluidfrom the process chamber 500. Furthermore, the solution addition unit590 can be configured to receive at least a portion of the targetconstituent extractor shown in FIGS. 21A-21B such that target-boundcomplexes can be transferred into a secondary container. Additionallyand/or alternatively, the solution addition unit 590 can be configuredto fluidically seal solution addition unit 590 from the bottom face 550by twisting the solution addition unit 590, completely removing solutionaddition unit 590 from the process chamber, adding a valve between thebottom face 550 and the solution addition unit 590, and/or can beotherwise configured with any other suitable subelement of the system.

The process chamber 200 can be optionally modified to increase theaggregation of target-bound complexes at a specific location of theprocess chamber (e.g., the interior sidewall 210 of the process chamber)to enhance isolation. In one variation, the process chamber 200 canencourage localization of target-bound complexes with at least one setof chamber morphologies (e.g., ridges, protrusions, grooves, etc.) thatare preferably fabricated using the press-fit process, but can befabricated using any other suitable manufacturing technique, (e.g.,micromachining, photolithography, 3D-printing, injection molding,laser-etching, roughening, scoring, tapping/threading, etc.).Furthermore, the set of chamber morphologies can be adhered to thesurface of the sidewall using any suitable method (e.g., lamination,heat-bonding, laser-bonding, anodic bonding), or otherwise joined to thesidewall. In a second variation, the interior surfaces can be treatedwith surface coatings (e.g., dip-coating, spray-coating,micropatterning, etc.), including chemical coatings, polymer coatings,and/or optical coatings. However, the interior sidewall 210 can includea single feature, or any other suitable number of features within theset of chamber morphologies configured to enable retention, orlocalization of a population of target-bound complexes within theprocess chamber at a collection region 230.

In one variation, as shown in FIG. 20A and FIG. 20B, the sidewall 210 ofthe process chamber 200 is patterned through a press fit process andincludes a set of protrusion features 235 (e.g., blocks, bumps,protrusions, positive space, etc.) that extend into the interior volumeof the process chamber. The set of protrusion features 235 can befabricated from any suitable material (e.g., polymer, metal, etc.). Theset of protrusion features preferably includes features with homogenousshape, dimension, and spacing, but can additionally and/or alternativelyinclude features with varying morphology, distribution, and/or anysuitable combination thereof. In a first example, as shown in FIG. 20A,the process chamber includes a set of triangular ridges 235, whereineach ridge has a ridge depth 236 (e.g., orthogonal to the side wall) anda between-ridge spacing 237 (e.g., along the sidewall), defined betweenadjacent ridges of the sidewall 210. Each ridge in the set of ridges isthe same dimension and spacing. In operation, as fluid is removed fromthe process chamber, target-bound complexes can settle into thebetween-ridge spacing 237, thus enabling non-target constituents to befully removed from the process chamber 200. To enhance the attraction oftarget-bound complexes to the sidewall 210 in aqueous systems, the idealratio of ridge depth 236 to between-ridge spacing 237 is within therange of 5:1 to 1:50. In another variation of the first example, asshown in FIG. 20B, the ridge depth 236 and ridge diagonal surface 238can be patterned in a step-like manner, where the ratio of the ridgedepth 236 to diagonal surface 238 can include ratios of (1:1, 1:10,1:100, 1:1000, and 1:10000). In a second example, the between-ridgespacing can decrease with corresponding height of the ridge on thesidewall, forming a patterned gradient of triangular ridges along thelongitudinal axis of the sidewall, wherein the density of triangularridges increases near the upper region of the process chamber.Preferably, the set of protrusion features 235 is continuous around theentirety of a specific height of the process chamber 200, but canalternatively be fabricated on a subregion of the sidewall 210. However,the protrusion features 235 of the sidewall 210 can be otherwiseconfigured, and can additionally and/or alternatively be used incombination with recessed features 245.

In a second variation, the sidewall 210 of the process chamber 200 ispatterned through a press fit process and includes a set of recessedfeatures 245 (e.g., holes, grooves, slots, negative space, etc.). Theset of recessed features 245 can include features of homogenous shape,dimension, and spacing, but can additionally and/or alternativelyinclude features with varying morphology, distribution, and/or anysuitable combination thereof. In an example, as shown in FIG. 20C, thesidewall 210 of the process chamber 200 can be texturized through ascoring process (e.g., manually or automated using a sharp edge, needle,etc.). The texture of the sidewall can include any suitable combinationof patterns, depth, width, or feature, such that the target-boundcomplexes have affinity for the lateral portions of the sidewall (e.g.,collection region 230) as fluid is removed from the vial. In a secondexample, as shown in FIG. 11, the sidewall includes a single recessedfeature 250, configured to retain a population of target-bound complexesat the collection region 230 (e.g., upper region of the processchamber). In a third example, the sidewalls of a 1.5 mL centrifuge tubecan be tapped, wherein a set of internal threads along the sidewall ofthe centrifuge tube can be formed using an M9-sized tap or any othersuitable tool (e.g., spiral flute tap, spiral point tap, pin, plug,etc.). The modified (e.g., threaded) sidewalls of the centrifuge tubecan then be used to efficiently capture target-bound complexes aroundthe entire circumference and upper section of the tube (e.g., due tosurface tension between the sample fluid and the set of internal threadsof the sidewall), However, the process chamber can be otherwise modifiedand/or configured. Preferably, the set of recessed features 245 iscontinuous around the entirety of a specific height of the processchamber 200, but can alternatively be fabricated on a subregion of thesidewall 210. However, the recessed features 245 of the sidewall 210 canbe otherwise configured, and can additionally and/or alternatively beused in combination with protrusion features 235.

Alternatively, in a third variation, the sidewall of the process chamberincludes features configured to move target-bound complexes away from(e.g., rather than towards) the sidewall (e.g., increasing the flowvelocity of the buoyant particles to a collection region). In oneexample, as shown in FIG. 1J, each of the sidewalls of a rectangularprocess chamber are fabricated at a set angle offset from vertical(e.g., 3° offset), enabling concentration of the buoyant particles inseparation steps described in Section 1. However, the sidewalls of theprocess chamber can be configured in any other suitable manner tomanipulate the movement and location of target-bound complexes.

Additionally or alternatively, in some variations, the process chamber200 can include a window 260 configured adjacent to the collectionregion 230, wherein the window comprises a planar substrate configuredto enable observation of target-bound complexes aggregated at thecollection region, as shown in FIG. 9A, or alternatively defines adetection volume configured to receive the population of target-boundcomplexes from the collection region and to interface with a detectormodule, as shown in FIG. 9B.

Additionally or alternatively, in some variations, the process chambercan comprise or be coupled to a dip tube 270 comprising a first end thatcouples to a fluid transfer element (e.g., a pipette tip) and a secondend configured inferior to the collection region at which buoyantsubstrates aggregate, in order to enable fluid transfer in the processchamber substantially without disturbance of the buoyant substrates. Inexamples, the dip tube can be incorporated into a lid of the processchamber, as shown in FIG. 12A, incorporated into a wall of the processchamber, as shown in FIG. 12B, be a modular component that interfaceswith the process chamber (e.g., by adhering to a wall of the processchamber with a membrane), and/or cooperate with the process chamber inany other suitable manner.

Additionally or alternatively, in some variations, the process chamber200 can comprise a cap 280 in communication with the collection region,wherein the cap 280 facilitates extraction of the population oftarget-bound complexes from the collection region of the processchamber. An additional example of a portion of the process chamber isshown in FIG. 13.

In related embodiments of a system 300 for separating and extracting apopulation of target-bound complexes from a sample, embodiments andvariations of which are shown in FIGS. 14-17C, the system 300 caninclude a process chamber 310 and an extraction apparatus 350 configuredto interface with the sample containing chamber 310 in differentoperation modes, in order to facilitate separation and/or enableextraction of a population of target-bound complexes from the sample.Alternative variations of these embodiments of the system 300 can,however, omit the process chamber 310.

In the related embodiments of the system 300, the process chamber 310functions to hold a sample and can additionally or alternativelyfunction to facilitate mixing of the target constituent of the samplewith a volume of buoyant substrates to produce a population oftarget-bound complexes. The process chamber 310 preferably has a closedend and an openable end (e.g., an end configured to be opened, apermanently open end, etc.) opposing the closed end, the process chamberconfigured to hold the sample having the target constituent andfacilitate binding of the target constituent to a set of substrates toproduce a set of buoyant target-bound complexes. The process chamber 310is preferably substantially rigid; however, the process chamber canalternatively be deformable (e.g., under compression, under tension,under torsion, etc.). In a specific example, the process chamber 310 iscomposed of plastic; however, the process chamber 310 can alternativelyinclude regions composed of one or more of: a ceramic material, ametallic material (e.g., to aid magnetic separation), and any othersuitable material. Furthermore, the process chamber 310 can additionallyor alternatively be configured in any other suitable manner.

2.2 System—Extraction Apparatus

In the related embodiments of the system 300, the extraction apparatus350 (i.e., target constituent extractor) functions to provide a surfaceor volume at which or into which the population of target-boundcomplexes can be transmitted, thereby facilitating extraction of thepopulation of target-bound complexes from a bulk volume of the sample.In the related embodiments, the extraction apparatus can compriseelements and/or be configured in any other suitable manner (e.g., withsurface treatments, protrusion features, recessed features) that enableretention of the population of target-bound complexes at the extractionapparatus 350, such as those described in the above Section 2.1. Theextraction apparatus 350 is preferably used to perform Block S140described in Section 1, but can be used to perform any other suitableportion of the method 100. Embodiments, variations, and/or examples ofthe extraction apparatus are further described in U.S. application Ser.No. 14/969,446, filed 15 Dec. 2015.

In a first variation, as shown in FIGS. 15A-15D and, the system 300′includes a sample containing chamber 310′ defining a volume forretention of the sample, within which the target constituent(s) of thesample can be combined with a volume of buoyant substrates that enableisolation of the target constituent from the sample in the form of apopulation of target-bound complexes. In the first variation, theextraction apparatus 350′ comprises a first portion 360′ including afrustoconical surface 362′ that defines a feeding region 363′ into anopening 364′ (e.g., having an inverted frustoconical surface thatopposes the frustoconical surface 362′) at a superior portion of thefirst portion 360′; and a second portion 370′ comprising a collectiontube 372′ that interacts with the opening 364′ of the feeding region363′ to receive particles of the population of target-bound complexesfrom the sample. In the first variation, the frustoconical surfacedefines a base region that interfaces with the sample volume in theprocess chamber 310′, and a concentration region (associated with theopening 364′) above and in opposition to the base region, wherein thefrustoconical surface defines a volume configured to concentrate the setof buoyant target-bound complexes at the concentration region of thefrustoconical surface. Furthermore, in the first variation, theextraction apparatus 350′ can include a separation zone having aninverted frustoconical surface 366′, in communication with theconcentration region of the frustoconical surface 362′ by a channel 365′that transmits the set of buoyant target-bound complexes into theseparation zone, for removal of the set of buoyant target-boundcomplexes from the concentration region. In the first variation, thesecond portion 370′ of the extraction apparatus 360′ can include aventing chamber 374′ configured to provide venting of the samplecontaining chamber 310′ during relative displacement between the samplecontaining chamber and the extraction apparatus 350′ for extraction ofthe population of target-bound complexes from the sample.

In the first variation, the first portion 360′ of the extractionapparatus 350′ having the frustoconical surface 362′ preferablyinterfaces with a surface of the sample fluid in a first configuration(e.g., a compressed configuration), as shown in FIG. 15B, such that thefeeding region 363′ receives fluid of the sample and enables feeding ofthe population of target-bound complexes toward the opening 364′ due tobuoyant separation. In the first configuration, the collection tube 372′is in position to receive the population of target-bound complexes asthey float in an inferior-to-superior direction, as shown in FIG. 15C.Finally, uncoupling of the second portion 370′ from the first portion360′ of the extraction apparatus 350′, as shown in FIG. 15D, enablesextraction of the population of target-bound complexes from the sample,by way of the collection tube 372′ of the second portion 370′ of theextraction apparatus 350′. In the first variation, the first portion360′ and the second portion 370′ of the extraction apparatus 350′ can bepositioned together and/or separately with respect to the processchamber 310′ of the process chamber 300′. Furthermore, in the firstvariation, the first portion 360′ and the second portion 370′ of theextraction apparatus 350′ can be displaced relative to each other and/orrelative to the process chamber 310′ by any one or more of a: screwmechanism, a sliding mechanism, a ratcheting mechanism, a magneticmechanism, and any other suitable mechanism. Furthermore, motion betweencomponents of the system 300′ of the first variation can be limited to aspecified range (e.g., with a tab), in association with phases of sampleprocessing to extract the target constituent from the sample.

In variations of the first variation, the opening 364′ of the feedingregion 363′ and the collection tube 372′ preferably mate with each otherin a complementary manner, and furthermore are flush with each other inorder to provide a sufficient seal that prevents particles of thepopulation of target-bound complexes from entering undesired portions ofthe process chamber 300′. As such, in a specific example where thefeeding region 363′ is associated with the inverted frustoconicalsurface 366′, as shown in FIG. 15A, the collection tube 372′ can becoupled to a second inverted frustoconical surface 376′ that iscomplementary to the inverted frustoconical surface 366′ coupled to thefeeding region 363′. However, the first portion 360′ and the secondportion 370′ can alternatively interact with each other in any othersuitable manner. Additionally or alternatively, in variations of thefirst variation, the process chamber 310′ can receive fluid (e.g.,buffer, sample) through a port (e.g., within the process chamber 310′and/or the extraction apparatus 350′, in order to facilitate delivery ofthe population of target-bound complexes toward the feeding region 363′and/or the collection tube 372′.

In another variation of the first variation, as shown in FIGS. 21A and21B, the extraction apparatus 350′ comprises a first portion 360′including a single frustoconical surface 362′ that defines a feedingregion 363′ into an opening 364′ that forms a continuous fluidic pathwayto the collection tube 372′, such that the buoyant particles can passfrom the sample containing chamber 380 to an upper chamber 385. Uponrelocation of the target-bound complexes to the upper chamber 385, theupper chamber 385 (e.g., a separate collection tube, secondarycontainer) can be closed and removed from the sample containing chamber380 (e.g., a process chamber), such that fluid in the upper chamber 385can be manipulated without any fluidic contact with the samplecontaining chamber 380. In one example, as shown in FIG. 21B, the firstportion 360′ can be configured as a collection plunger that operates incoordination with a valve slit 390 coupled to a vent path 392, and canoptionally include a seal 395 that allows coupling of the extractionapparatus 350′ to the sample containing chamber 380. When the upperchamber 385 is coupled to the sample containing chamber 380, thecollection plunger can be arranged in a first configuration (e.g.,lowered configuration) to enable target-bound complexes to travel intothe upper chamber 385, wherein the valve slit 390 is closed and air isunable to pass through the vent path 392. Once transfer is complete, theupper chamber 385 can be secured with the separator lid 398 and removedfrom the sample containing chamber 380, thus opening the vent path 392to allow air to pass through the valve slit such that the fluid in theupper chamber 385 stays in the upper chamber 395. In one variation, uponremoval of the upper chamber from the process chamber, the upper chambercan be fluidly sealed using a vertically-aligned plug (e.g., valve, pin,etc.) inserted at the base of the upper chamber, thus allowing theseparator lid to be removed from the top surface of the upper chamberfor enriched sample processing. However, the extraction apparatus 350′can be otherwise secured and or/configured to enable manipulation anddownstream processing of the enriched sample contained in the upperchamber after extraction.

In an alternative variation of the first variation, the extractionapparatus 350′ can omit a second portion 370′, where buoyant particlesare configured to be transmitted from the process chamber 310′ into theinverted frustoconical surface 366′ of the first portion 360′ (accordingto one or more of the methods describe in Block S140 above), andextracted from the inverted frustoconical surface 366′ (according tomethods described above).

Furthermore, while frustoconical surfaces are described in relation tothe extraction apparatuses 350, 350′, variations of the extractionapparatus can alternatively define any other suitable surface (e.g.,broadening surface, narrowing surface) configured to facilitateconcentration and/or extraction of buoyant particles from the processchamber.

In a second variation, as shown in FIGS. 16A-16F, the system 300″includes a process chamber 310″ defining a volume for retention of thesample, within which the target constituent(s) of the sample can becombined with a volume of buoyant substrates that enable isolation ofthe target constituent from the sample in the form of a population oftarget-bound complexes. In the second variation, the extractionapparatus 350′ comprises a first portion 360″ including a hydrophilicregion 367″ and a hydrophobic region 368″ at a surface configured tointerface with a surface of sample fluid within the sample containingchamber 310″. In the second variation, the hydrophilic region 367″ issurrounded by the hydrophobic region 368″, in order to define an area atwhich the population of target-bound complexes can be retained forextraction from the sample containing chamber 310″. The size of thehydrophilic region 367″ in the second variation can further be adjustedor adjustable to provide an area sufficient in size to retain a desiredportion of the population of target-bound complexes at the first portion360″ of the extraction apparatus 350″. In the second variation, thehydrophilic region 367″ can be located at an external surface of thefirst portion 360″ that interfaces with fluid of the sample in thesample containing chamber 310″, or can alternatively be located at aninterior surface of the first portion 360″, and accessible through anopening into the first portion 360″ that also enables venting of theprocess chamber during extraction. Additionally or alternatively, thehydrophilic region 367″ can be composed of the same material as thefirst portion 360″ of the extraction apparatus 350″, wherein a coatingof hydrophobic material provides the hydrophobic region 368″ andisolates the hydrophilic region 367″ to a desired area of the extractionapparatus. Furthermore, the hydrophilic region 367″ can comprise anysuitable morphology (e.g., convex morphology, textured morphology) thatenhances contact between the hydrophilic region 367″ and fluid of thesample in the sample-containing chamber 310″.

In the second variation, the first portion 360″ of the extractionapparatus 350″ preferably interfaces with a surface of the sample fluidin a first operation mode (e.g., a compressed configuration), as shownin FIG. 16D such that the hydrophilic region 367″ interfaces with thepopulation of target-bound complexes due to buoyant separation in thesample. Upon displacement of the first portion 360″ of the extractionapparatus 350″ away from the sample container 310″ in a second operationmode, as shown in FIG. 16E, the population of target-bound complexes,coupled to the hydrophilic region 367″ by adhesion forces, is extractedfrom the sample containing portion 310″ of the process chamber 300″.Finally, uncoupling of the first portion 360″ from the process chamber310″, as shown in FIG. 16F, enables extraction of the population oftarget-bound complexes from the sample. In the second variation, thepopulation of target-bound complexes can then be retrieved (e.g., bypipetting) from the hydrophilic region 367″ of the first portion 360″ ofthe extraction apparatus. Similar to the first variation of the processchamber 300′, in the second variation, the first portion 360″ of theextraction apparatus 350′ can be displaced relative to the samplecontaining chamber 310″ by any one or more of: a screw mechanism, asliding mechanism, a ratcheting mechanism, a magnetic mechanism, and anyother suitable mechanism. Furthermore, motion between components of theprocess chamber 300′ of the first variation can be limited to aspecified range (e.g., with a tab), in association with phases of sampleprocessing to extract the target constituent from the sample.

In a third variation, as shown in FIGS. 17A-17C the process chamber300′″ includes a sample containing chamber 310′″ defining a volume forretention of the sample, within which the target constituent(s) of thesample can be combined with a volume of buoyant substrates that enableisolation of the target constituent from the sample in the form of apopulation of target-bound complexes. In the first variation, theextraction apparatus 350′″ comprises a first portion 360′″ including afrustoconical surface 362′″ that defines a feeding region 363′″ into anopening 364′″ at a superior portion of the first portion 360′″; and asecond portion 370′″ comprising a valve 376′″ (e.g., a ball valve, othervalve, etc.) that interfaces with the opening 364′″ in permittingcontrolled passage of the population of target-bound complexes into acollection region 378′″ of the second portion 370′″. In the thirdvariation, the second portion 370″″ includes a venting chamber 374″″having an inverted frustoconical surface opposing the frustoconicalsurface 362′″, configured to provide venting of the sample containingchamber 310″″ during relative displacement between the sample containingchamber and the extraction apparatus 350″″ for extraction of thepopulation of target-bound complexes from the sample.

In the third variation, the first portion 360″″ of the extractionapparatus 350′″ preferably interfaces with a surface of the sample fluidin a first configuration (e.g., a compressed configuration, prior toaddition of fluid into the venting chamber 374′″, etc.), as shown inFIG. 17B, such that the feeding region 363′″ receives fluid of thesample and enables feeding of the population of target-bound complexestoward the opening 364′″ due to buoyant separation. In the firstconfiguration, the valve 376′″ is displaced by fluid of the sample at asuperior region of the process chamber 310′″, thus allowing passage ofthe population of target-bound complexes, through the opening 364′″ andinto the venting chamber 374′″ of the second portion 370′″ of theextraction apparatus 350′″, as shown in FIG. 17C. Finally, uncoupling ofthe extraction apparatus 350′″ from the sample containing chamber 310′″enables extraction of the population of target-bound complexes from thesample. In the third variation, a specific volume of fluid (e.g., 100μL, 200 μL) can be configured to pass into the second portion 370′″ ofthe extraction apparatus 350′″ upon transmission of the extractionapparatus 350′″ into the first configuration within the process chamber(e.g., by linear displacement, by screwing the extraction apparatus andthe process chamber relative to each other, etc.). The specific volumeof fluid can be limited by a physical feature (e.g., notch) of theprocess chamber 300′″ that physically stops fluid passage past the valve376′″ once the set volume of fluid has been obtained. However, thespecific volume of fluid can be limited in any other suitable manner.

Similar to the first variation of the system 300, in the thirdvariation, the first portion 360′″ and the second portion 370′″ of theextraction apparatus 350′″ can be displaced relative to the samplecontaining chamber 310′″ by any one or more of a: screw mechanism, asliding mechanism, a ratcheting mechanism, a magnetic mechanism, and anyother suitable mechanism. Furthermore, motion between components of thesystem 300′″ of the first variation can be limited to a specified range(e.g., with a tab), in association with phases of sample processing toextract the target constituent from the sample. The system 300 can,however, comprise any suitable combination of the above variationsand/or any other suitable process chamber for processing a sample andenabling extraction of a target constituent from the sample.

Additionally and/or alternatively, variations of the process chamber andor/the extraction apparatus 350, 350′ can be integrated into anautomated system for removal and manipulation of sample volumes.Furthermore, modification of the sidewalls of any portion of theextraction apparatus (e.g., process chamber 310, first portion 360,collection tube 372, second portion 370, etc.) can allow target-boundcomplexes to be transferred in a predictable manner, such that automatedsystem can reliably extract fluid target-bound complexes and/orsurrounding fluid. In one variation, using microbubbles substrateparticles in a microwell plate (e.g., 96 to 1536 well plates),separation of target-bound complexes from the total sample volume can beperformed across multiple plates simultaneously and in parallel, asopposed to in series (as is done with magnetic particle manipulation),increasing throughput and minimizing the number of steps for separation.In one example, parallel processing (e.g., separation and extraction oftarget-bound complexes from multiple samples simultaneously) can beachieved using an automated system by stacking microwell plates. Oncetarget-bound complexes have been transferred to the upper portion of themicrowells (e.g., by flotation, at a specific time after combination),multiple plates can be simultaneously (e.g., via automation)re-positioned for targeted removal of substrate-bound complexes proximalthe upper surface of the sample fluid (e.g., at the meniscus), and/orthe non-target fluid (e.g., supernatant) proximal the lower surface ofthe sample fluid (e.g., below the meniscus). However, automation ofseparation and extraction steps can be performed using any suitablecombination of subcomponents of the system, in any other suitablemanner.

As such, the process chamber systems 200, 300 are preferably configuredto perform at least a portion of the method 100 described in Section 1above; however, the process chamber 200 can additionally oralternatively be configured to perform any other suitable method.

2.3 System—Alternative Variations

Alternative variations of the process chambers 200, 300 can, however,comprise any other suitable container (e.g., fluid receiving and/ordistributing bag). For instance, in one variation aspects of theseparation and/or extraction elements of the systems described above canbe incorporated into a bag-type form factor (e.g., blood transfusionbag, leukopheresis bag, cell collection bag, etc.), where generating apopulation of target-bound complexes is performed, and the bag-type formfactor facilitates separation and/or extraction of the population oftarget-bound complexes from a sample volume. Furthermore, bag-type formfactors can be used in closed-system operation, allowing highersterility for sensitive samples. In one such variation, as shown inFIGS. 18A and 18B, the bag 400 can include a set of stems 410incorporated into the bag, that function to enable accessing of the bagfor filling and emptying of contents of the bag 400. The stems 410 canadditionally or alternatively function to facilitate testing of a sample(e.g., blood type cross-matching, creation of a closed system forsterile extraction, sample enrichment, cell signaling, etc.). Inoperation, the set of stems 410 can thus allow buoyant particles to bereceived into the bag 400, to interact with and bind to sampleconstituents of interest, and then to be separated, for instance, usinga sealing mechanism (e.g., heat sealing mechanism) to seal portions ofthe bag 400 containing the buoyant particles from portions of the bag400 that are substantially void of the buoyant particles.

In a first variation, as shown in FIG. 18A, the bag 400′ can include aset of inferiorly located stems 410′, such that draining of the bag 400′through one or more of the stems 410′ causes a population oftarget-bound complexes within the bag to enter the stems 410′, with asmall amount of sample fluid, for extraction. Additionally oralternatively, in the first variation, one or more stems 410′ of the bag400′ can be sealed off and/or removed from the bag 400′, therebyenabling extraction of the population of target-bound complexes from thebulk sample.

In a second variation, as shown in FIG. 18B, the bag 400″ can include aset of superiorly located stems 410″, such that delivery of thepopulation of target-bound complexes into the stems 410″ (e.g., throughcompression of the bag, through buoyant forces, etc.) allows thepopulation of target-bound complexes to be separated from the bulksample volume in the bag 400″. Additionally or alternatively, in thesecond variation, one or more stems 410″ of the bag 400′ can be sealedoff and/or removed from the bag 400″, thereby enabling extraction of thepopulation of target-bound complexes from the bulk sample.

In still alternative embodiments, a process chamber 600, as shown inFIG. 19A, can include an outlet 610, wherein the outlet 610 can includeone or more of: a puncturable diaphragm, a luer lock, a valve, and/orany other suitable outlet. The outlet can thus allow sample fluid and/ornon-buoyant particles to be removed from the process chamber 600,without disturbing a region of separated buoyant particles. Invariations of these alternative embodiments, the outlet 610 can thusfacilitate one or more of: negative separation (e.g., to removenon-target constituents from the sample) and positive separation (e.g.,to remove target constituents from the sample). Additionally oralternatively, the process chamber 600 and/or outlet 610 can facilitateperformance of chemistry on buoyant particles (e.g., in a two-phasesolvent system). Additionally or alternatively, the process chamber 600and/or outlet 610 can facilitate removal of compromised buoyantparticles (e.g., broken buoyant particles) from the bulk sample volume.However, the chamber 600 and/or outlet 610 can facilitate any othersuitable operation in relation to the population of target-boundcomplexes.

In one variation, the process chamber 600′ can include an inferiorlylocated outlet 610′, such that draining of the process chamber 600′through the outlet 610′ causes a population of target-bound complexeswithin the process chamber 600′ to enter an inferior region of theprocess chamber 600′ (in the orientation shown in FIG. 19B), with asmall amount of sample fluid, for extraction. Additionally oralternatively, in the first variation, the inferior region 620′ of theprocess chamber 600′ can be configured to separate from the remainder ofthe process chamber 600′, as shown in FIG. 19B, thereby enablingextraction of the population of target-bound complexes from the bulksample.

In an alternative variation, as shown in FIG. 19C, the process chamber600″ can include a superiorly located outlet 610″, wherein delivery ofthe population of target-bound complexes into a superior region 620″ ofthe process chamber 600′ (e.g., through buoyant forces, etc.) allows thepopulation of target-bound complexes to be separated from the bulksample volume in the process chamber 600″. Additionally oralternatively, in the second variation, the superior region 620″ of theprocess chamber 600″ can be sealed off and/or removed from the remainderof the process chamber 600″, thereby enabling extraction of thepopulation of target-bound complexes from the bulk sample. Additionallyor alternatively, the process chamber 600″ of this variation can beinverted, non-buoyant components of the sample can be drained fromoutlet 610″, and then the process chamber 600″ can then be reverted to anon-inverted orientation, whereby the population of target-boundcomplexes remains in the superior region 620″ of the process chamber600″ due to surface tension between fluid coupled to the population oftarget-bound complexes and the wall of the superior region 620″ of theprocess chamber. The superior region 620″ of the process chamber 600″can then be sealed off and/or removed from the remainder of the processchamber 600″, thereby enabling extraction of the population oftarget-bound complexes from the bulk sample.

Additionally or alternatively, variations of the process chambers 200,300 can be composed of or otherwise include metallic regions (e.g.,magnetic regions, ferromagnetic regions) configured to facilitatemagnetic separation of the target constituent(s) of the sample accordingto methods described in Section 1 above. For instance,magnetic/ferromagnetic regions can facilitate formation of stray fieldsand/or directed magnetic fields that enable magnetic separation ofsample components in addition to or in alternative to buoyancy-basedseparation methods.

The FIGURES illustrate the architecture, functionality and operation ofpossible implementations of systems, methods and computer programproducts according to preferred embodiments, example configurations, andvariations thereof. In this regard, each block in the flowchart or blockdiagrams may represent a module, segment, step, or portion of code,which comprises one or more executable instructions for implementing thespecified logical function(s). It should also be noted that, in somealternative implementations, the functions noted in the block can occurout of the order noted in the FIGURES. For example, two blocks shown insuccession may, in fact, be executed substantially concurrently, or theblocks may sometimes be executed in the reverse order, depending uponthe functionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts, or combinations of special purpose hardware andcomputer instructions.

The method 100 and/or system 200 of the preferred embodiment can beembodied and/or implemented at least in part as machine configured toreceive a computer-readable medium storing computer-readableinstructions. The instructions are preferably executed bycomputer-executable components preferably integrated with the system andone or more portions of the processor and/or analysis engine. Thecomputer-readable medium can be stored in the cloud and/or on anysuitable computer-readable media such as RAMs, ROMs, flash memory,EEPROMs, optical devices (CD or DVD), hard drives, floppy drives, or anysuitable device. The computer-executable component is preferably ageneral or application specific processor, but any suitable dedicatedhardware or hardware/firmware combination device can alternatively oradditionally execute the instructions.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

We claim:
 1. A method for buoyant separation of a set of targetconstituents of a sample, the method comprising: at a vessel, receiving:the sample comprising the set of target constituents and a set ofnon-target constituents, wherein bulk fluid of the sample has a firstdensity; a volume of buoyant particles having a second density, thesecond density less than the first density, wherein the buoyantparticles are configured to bind with at least a portion of the set oftarget constituents thereby producing a set of target-bound complexes,wherein the volume of buoyant particles and the set of targetconstituents are received at the vessel at a ratio between 1:2 and 5:1;and concentrating the set of target-bound complexes at a superior regionof the vessel, thereby producing the buoyant separation.
 2. The methodof claim 1, further comprising a mixing process, wherein the mixingprocess is configured to increase a speed associated with concentratingthe set of target-bound complexes at the superior region.
 3. The methodof claim 2, wherein the mixing process is an end-over-end mixingprocess.
 4. The method of claim 1, wherein each of the set of buoyantparticles is a glass microbubble.
 5. The method of claim 4, wherein thesecond density is between 0.4 and 0.8 grams-per-centimeter-cubed.
 6. Themethod of claim 1, further comprising processing the set of non-targetconstituents in the vessel.
 7. The method of claim 1, further comprisingprocessing the set of target-bound complexes in a second vessel.
 8. Themethod of claim 1, further comprising processing the set of target-boundcomplexes in the vessel.
 9. The method of claim 8, further comprisingdraining the set of non-target constituents from the vessel prior toprocessing the set of target-bound complexes in the vessel.
 10. Themethod of claim 9, wherein the vessel comprises a valve configured todrain the set of non-target constituents from an outlet of the vessel.11. The method of claim 10, wherein the outlet is arranged at aninferior-most region of the vessel.
 12. The method of claim 1, whereinthe ratio is between 1:1 and 2.5:1.
 13. A system for separation of a setof target constituents of a sample, the system comprising: a volume ofbuoyant particles having a first density; a vessel defining a cavity,wherein the vessel: receives the sample, wherein the sample comprisesthe set of target constituents and a set of non-target constituents,wherein bulk fluid of the sample has a second density, the seconddensity greater than the first density; receives the volume of buoyantparticles, wherein the buoyant particles are configured to bind with atleast a portion of the set of target constituents, thereby producing aset of target-bound complexes, and wherein the volume of buoyantparticles and the set of target constituents are received at the vesselat a ratio between 1:2 and 5:1; and concentrates the set of target-boundcomplexes at a superior region of the vessel, thereby producing thebuoyant separation.
 14. The system of claim 13, wherein the vesselcomprises a valve.
 15. The system of claim 14, wherein the valve isarranged at an inferior region of the vessel.
 16. The system of claim15, wherein the valve is configured to drain the set of non-targetconstituents from the inferior region of the vessel after the set oftarget-bound complexes have been concentrated at the superior region ofthe vessel.
 17. The system of claim 13, wherein each of the set ofbuoyant particles is a glass microbubble.
 18. The system of claim 17,wherein the first density is between 0.4 and 0.8grams-per-centimeter-cubed.
 19. The system of claim 13, wherein theratio is between 1:1 and 2.5:1.
 20. The system of claim 13, wherein thecavity is a single cavity.